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Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase

Nature Biotechnology volume 20, pages 1135–1139 (2002)Cite this article

Abstract

We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12–C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3–C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.

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Figure 1: Maximum turnover rates (mol substrate/min/mol enzyme) for P450 BM-3 wild type and 139-3.
Figure 2: Maximum rates reported for alkane hydroxylation by alkane monooxygenases.
Figure 3: Optical spectra for 139-3 and wild-type P450 BM-3.
Figure 4: Positions of the amino acid substitutions in P450 BM-3 mutant 139-3.

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References

  1. Chen, H.Y., Schlecht, S., Semple, T.C. & Hartwig, J.F. Thermal, catalytic, regiospecific functionalization of alkanes. Science 287, 1995–1997 (2000).

    Article  CAS  Google Scholar 

  2. Hartmann, M. & Ernst, S. Selective oxidations of linear alkanes with molecular oxygen on molecular sieve catalysts—a breakthrough? Angew. Chem. Intl. Edn. 39, 888–890 (2000).

    Article  CAS  Google Scholar 

  3. Thomas, J.M., Raja, R., Sankar, G. & Bell, R.G. Molecular sieve catalysts for the regioselective and shape-selective oxyfunctionalization of alkanes in air. Accounts Chem. Res. 34, 191–200 (2001).

    Article  CAS  Google Scholar 

  4. Ashraf, W., Mihdhir, A. & Murrell, J.C. Bacterial oxidation of propane. FEMS Microbiol. Lett. 122, 1–6 (1994).

    Article  CAS  Google Scholar 

  5. Watkinson, R.J. & Morgan, P. Physiology of aliphatic hydrocarbon-degrading microorganisms. Biodegradation 1, 79–92 (1990).

    Article  CAS  Google Scholar 

  6. Leadbetter, E.R. & Foster, J.W. Incorporation of molecular oxygen in bacterial cells utilizing hydrocarbons for growth. Nature 184, 1428–1429 (1959).

    Article  CAS  Google Scholar 

  7. Scheller, U., Zimmer, T., Kargel, E. & Schunck, W.H. Characterization of the n-alkane and fatty acid hydroxylating cytochrome P450 forms 52A3 and 52A4. Arch. Biochem. Biophys. 328, 245–254 (1996).

    Article  CAS  Google Scholar 

  8. Stevenson, J.A., Westlake, A.C.G., Whittock, C. & Wong, L.L. The catalytic oxidation of linear and branched alkanes by cytochrome P450(cam). J. Am. Chem. Soc. 118, 12846–12847 (1996).

    Article  CAS  Google Scholar 

  9. Fox, B.G., Froland, W.A., Jollie, D.R. & Lipscomb, J.D. Methane monooxygenase from Methylosinus trichosporium Ob3b. Methods Enzymol. 188, 191–202 (1990).

    Article  CAS  Google Scholar 

  10. Fisher, M.B., Zheng, Y.M. & Rettie, A.E. Positional specificity of rabbit CYP4B1 for omega-hydroxylation of short-medium chain fatty acids and hydrocarbons. Biochem. Biophys. Res. Com. 248, 352–355 (1998).

    Article  CAS  Google Scholar 

  11. Benson, S., Oppici, M., Shapiro, J. & Fennewald, M. Regulation of membrane peptides by Pseudomonas plasmid alk regulon. J. Bacteriol. 140, 754–762 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Schmid, A. et al. Industrial biocatalysis today and tomorrow. Nature 409, 258–268 (2001).

    Article  CAS  Google Scholar 

  13. Boddupalli, S.S., Estabrook, R.W. & Peterson, J.A. Fatty-acid monooxygenation by cytochrome P450 BM-3. J. Biol. Chem. 265, 4233–4239 (1990).

    CAS  PubMed  Google Scholar 

  14. Narhi, L.O. & Fulco, A.J. Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P450 monooxygenase induced by barbiturates in Bacillus megaterium. J. Biol. Chem. 261, 7160–7169 (1986).

    CAS  PubMed  Google Scholar 

  15. Miura, Y. & Fulco, A.J. ω-1, ω-2, and ω-3 hydroxylation of long-chain fatty acids, amides, and alcohols by a soluble enzyme system from Bacillus megaterium. Biochim. Biophys. Acta 338, 305–317 (1975).

    Article  Google Scholar 

  16. Farinas, E.T., Schwaneberg, U., Glieder, A. & Arnold, F.H. Directed evolution of a cytochrome P450 monooxygenase for alkane oxidation. Adv. Synth. Catal. 343, 601–606 (2001).

    Article  CAS  Google Scholar 

  17. Munro, A.W., Lindsay, J.G. & Coggins, J.R. Alkane metabolism by cytochrome P450 BM3. Biochem. Soc. T. 21, 412S (1993).

    Article  CAS  Google Scholar 

  18. Schwaneberg, U., Otey, C., Cirino, P.C., Farinas, E. & Arnold, F.H. Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Escherichia coli. J. Biomol. Screen 6, 111–117 (2001).

    CAS  PubMed  Google Scholar 

  19. Schwaneberg, U., Schmidt-Dannert, C., Schmitt, J. & Schmid, R.D. A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A. Anal. Biochem. 269, 359–366 (1999).

    Article  CAS  Google Scholar 

  20. Shanklin, J., Achim, C., Schmidt, H., Fox, B.G. & Munck, E. Mossbauer studies of alkane omega-hydroxylase: evidence for a diiron cluster in an integral-membrane enzyme. Proc. Natl. Acad. Sci. USA 94, 2981–2986 (1997).

    Article  CAS  Google Scholar 

  21. Fox, B.G., Froland, W.A., Dege, J.E. & Lipscomb, J.D. Methane monooxygenase from Methylosinus trichosporium Ob3b—purification and properties of a 3-component system with high specific activity from a Type-Ii methanotroph. J. Biol. Chem. 264, 10023–10033 (1989).

    CAS  PubMed  Google Scholar 

  22. Tonge, G.M., Harrison, D.E. & Higgins, I.J. Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b. Biochem. J. 161, 333–344 (1977).

    Article  CAS  Google Scholar 

  23. Green, J. & Dalton, H. Substrate-specificity of soluble methane monooxygenase—mechanistic implications. J. Biol. Chem. 264, 17698–17703 (1989).

    CAS  PubMed  Google Scholar 

  24. Murrell, J.C., Gilbert, B. & McDonald, I.R. Molecular biology and regulation of methane monooxygenase. Arch. Microbiol. 173, 325–332 (2000).

    Article  CAS  Google Scholar 

  25. Staijen, I.E., van Beilen, J.B. & Witholt, B. Expression, stability and performance of the three-component alkane mono-oxygenase of Pseudomonas oleovorans in Escherichia coli. Eur. J. Biochem. 267, 1957–1965 (2000).

    Article  CAS  Google Scholar 

  26. Zimmer, T., Ohkuma, M., Ohta, A., Takagi, M. & Schunck, W.H. The CYP52 multigene family of Candida maltosa encodes functionally diverse n-alkane-inducible cytochromes P450. Biochem. Biophys. Res. Commun. 224, 784–789 (1996).

    Article  CAS  Google Scholar 

  27. Groves, J.T. & Han, Y.Z. Models and mechanisms of cytochrome P450 action. in Cytochrome P450: Structure, Mechanism, and Biochemistry 2nd Edn (ed. Ortiz de Montellano, P.R.) 3–48 (Plenum Press, New York, 1995).

    Chapter  Google Scholar 

  28. Haines, D.C., Tomchick, D.R., Machius, M. & Peterson, J.A. Pivotal role of water in the mechanism of P450BM-3. Biochemistry 40, 13456–13465 (2001).

    Article  CAS  Google Scholar 

  29. Li, H.Y. & Poulos, T.L. The structure of the cytochrome P450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid. Nat. Struct. Biol. 4, 140–146 (1997).

    Article  CAS  Google Scholar 

  30. Paulsen, M.D. & Ornstein, R.L. Dramatic differences in the motions of the mouth of open and closed cytochrome P450BM-3 by molecular dynamics simulations. Proteins 21, 237–243 (1995).

    Article  CAS  Google Scholar 

  31. Chang, Y.T. & Loew, G. Homology modeling, molecular dynamics simulations, and analysis of CYP119, a P450 enzyme from extreme acidothermophilic archaeon Sulfolobus solfataricus. Biochemistry 39, 2484–2498 (2000).

    Article  CAS  Google Scholar 

  32. Noble, M.A. et al. Roles of key active-site residues in flavocytochrome P450 BM3. Biochem. J. 339, 371–379 (1999).

    Article  CAS  Google Scholar 

  33. Appel, D., Lutz-Wahl, S., Fischer, P., Schwaneberg, U. & Schmid, R.D. A P450BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes. J. Biotechnol. 88, 167–171 (2001).

    Article  CAS  Google Scholar 

  34. Joo, H., Lin, Z.L. & Arnold, F.H. Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 399, 670–673 (1999).

    Article  CAS  Google Scholar 

  35. Cirino, P.C. & Arnold, F.H. Regioselectivity and activity of cytochrome P450 BM-3 and mutant F87A in reactions driven by hydrogen peroxide. Adv. Synth. Catal. in press (2002).

  36. Reipa, V., Mayhew, M.P. & Vilker, V.L. A direct electrode-driven P450 cycle for biocatalysis. Proc. Natl. Acad. Sci. USA 94, 13554–13558 (1997).

    Article  CAS  Google Scholar 

  37. Mathys, R.G., Schmid, A. & Witholt, B. Integrated two-liquid phase bioconversion and product-recovery processes for the oxidation of alkanes: process design and economic evaluation. Biotechnol. Bioeng. 64, 459–477 (1999).

    Article  CAS  Google Scholar 

  38. Omura, T. & Sato, R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370–2378 (1964).

    CAS  PubMed  Google Scholar 

  39. Zhao, H.M., Giver, L., Shao, Z.X., Affholter, J.A. & Arnold, F.H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258–261 (1998).

    Article  CAS  Google Scholar 

  40. Yeom, H. & Sligar, S.G. Oxygen activation by cytochrome P450BM-3: effects of mutating an active site acidic residue. Arch. Biochem. Biophys. 337, 209–216 (1997).

    Article  CAS  Google Scholar 

  41. Modi, S. et al. Nmr-studies of substrate-binding to cytochrome P450 (BM3): comparisons to cytochrome P450 (cam). Biochemistry 34, 8982–8988 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the National Science Foundation, the Max Kade Foundation (fellowship to A.G.), and by Maxygen. The authors also thank Nathan Dalleska for his assistance with GC/MS, and Ulrich Schwaneberg (International University, Bremen, Germany), Tom Poulos (University of California, Irvine, USA), and Andrew Munro (University of Leicester, UK) for helpful discussions. Correspondence and requests for material should be addressed to F.H.A.

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Author notes

  1. Anton Glieder and Edgardo T. Farinas: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Biotechnology, Technical University of Graz, Petersgasse 12, Graz, A-8010, Austria

    Anton Glieder

  2. Division of Chemistry and Chemical Engineering, California Institute of Technology, MC 210-41, Pasadena, 91125, CA, USA

    Edgardo T. Farinas & Frances H. Arnold

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  1. Anton Glieder
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Correspondence to Frances H. Arnold.

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Glieder, A., Farinas, E. & Arnold, F. Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase. Nat Biotechnol 20, 1135–1139 (2002). https://doi.org/10.1038/nbt744

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